Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

Andrew E. Nixon, Steven M. Firestine, Frank G. Salinas, Stephen J. Benkovic

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The generation of enzymes to catalyze specific reactions is one of the more challenging problems facing protein engineers. Structural similarities between the enzyme scytalone dehydratase with nuclear transport factor 2 (NTF2) suggested the potential for NTF2 to be re-engineered into a scytalone dehydratase-like enzyme. We introduced four key catalytic residues into NTF2 to create a scytalone dehydratase-like active site. A C-terminal helix found in scytalone dehydratase but absent in NTF2 also was added. Mutant NTF2 proteins were tested for catalytic activity by using a spectroscopic assay. One of the engineered enzymes exhibited catalytic activity with minimal k(cat) and K(m) values of 0.125 min-1 and 800 μM, respectively. This level of catalytic activity represents minimally a 150-fold improvement in activity over the background rate for substrate dehydration and a dramatic step forward from the catalytically inert parent NTF2. This work represents one of the few examples of converting a protein scaffold into an enzyme, outside those arising from the induction of catalytic activity into antibodies.

Original languageEnglish (US)
Pages (from-to)3568-3571
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number7
DOIs
StatePublished - Mar 30 1999

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scytalone dehydratase
Cell Nucleus Active Transport
Enzymes
Proteins
Dehydration
Catalytic Domain

All Science Journal Classification (ASJC) codes

  • General

Cite this

Nixon, Andrew E. ; Firestine, Steven M. ; Salinas, Frank G. ; Benkovic, Stephen J. / Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold. In: Proceedings of the National Academy of Sciences of the United States of America. 1999 ; Vol. 96, No. 7. pp. 3568-3571.
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Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold. / Nixon, Andrew E.; Firestine, Steven M.; Salinas, Frank G.; Benkovic, Stephen J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 7, 30.03.1999, p. 3568-3571.

Research output: Contribution to journalArticle

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